Mercury is an element of great pharmacological and toxicological importance. It reacts with sulfhydryl groups on proteins to form mercaptides. Mercuric mercury (Hg2+), a form that shows primarily epithelial toxicity, can inhibit Na+/K(+)-ATPase at low concentration, but its molecular target site on the protein is not known. To investigate the interaction of Hg2+ with Na+/K(+)-ATPase, we studied the inhibition of Na+/K+ pump activity by inorganic mercury (HgCl2) in Xenopus laevis oocytes expressing wild-type and mutant forms of Na+/K(+)-ATPase. Na+/K+ pump potassium-activated current was inhibited with first-order kinetics (Kon = 7 x 10(3) M-1.sec-1) and an estimated Kd of < or = 170 nM. To study the hypothesis that the cysteine (C113) of the first transmembrane segment of the alpha subunit participates in a Hg2+ binding site, we investigated the inhibition of Na+/K+ pump activity produced by a 1-min exposure to 5 microM HgCl2. Wild-type and C113S and C113Y mutant Na+/K+ pumps were inhibited by 43 +/- 7%, 12 +/- 2%, and 5 +/- 3%, respectively. Because C113 is a component of the cardiac steroid binding site, we studied the interaction of mercury with strophanthidin by exposing oocytes for 2 min to 5 microM HgCl2 in the presence or absence of 50 microM strophanthidin. Strophanthidin reduced the inhibition by mercury from 68 +/- 5% to 30 +/- 7%. Based on the position of C113 in the first transmembrane segment, these results suggest that Hg2+ binding to C113 from the extracellular side is one of the mechanisms by which mercury inhibits Na+/K(+)-ATPase.